Rapid Modeling of Immunosuppressive Mechanisms in GEMMs with CRISPR/Cas9
Megan L. Burger1, Santiago Naranjo1, Tamina Kienka1, Tyler Jacks1,2
1Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA; 2Howard Hughes Medical Institute, Cambridge, MA
One explanation for tumor resistance to checkpoint blockade is immunosuppression active in the tumor microenvironment. Several factors have been implicated in mediating this effect, including recruitment of suppressive immune cell types, including T regulatory cells, and expression of immunosuppressive molecules, such as PD-L1 and indoleamine-2,3-dioxygenase (IDO). Combination therapies aimed at breaking down these immunosuppressive barriers hold promise for improving the efficacy of checkpoint immunotherapies. To facilitate further study of mechanisms of immunosuppression in a physiological context, we have developed a CRISPR/Cas9 approach to promote tumor-specific activation of immunosuppressive factors in autochthonous mouse models of cancer. Delivery of a lentivirus expressing a truncated sgRNA and a ‘SAM’ transcriptional activator complex into Cas9-expressing cell lines results in robust activation of immunosuppressive factors, including PD-L1 and IDO, at both the mRNA and protein level. Introduction of a PD-L1 virus that also contains Cre recombinase into the lungs of KrasLSL-G12D; p53fl/fl; Rosa26Cas9/+ mice results in tumor-specific PD-L1 activation that is maintained through late tumor stages. We hope this system will negate the need for laborious genetic crosses, allowing for the rapid interrogation of both known and candidate immunosuppressive factors in mouse models that faithfully recapitulate the complex tumor microenvironment.